Nosocomial infections are infections that are a result of treatment in a hospital or a healthcare service unit. Infections are considered nosocomial if they first appear 48 hours or more after hospital admission or within 30 days after discharge. This type of infection is also known as a hospital-acquired infection (or, in generic terms, healthcare-associated infection). In the United States, the Center for Disease Control and Prevention estimates that roughly 1.7 million hospital-associated infections, from all types of microorganism, including bacteria, combined, cause or contribute to 99,000 deaths each year. In Europe, where hospital surveys have been conducted, the category of Gram-negative infections are estimated to account for two-thirds of the 25,000 deaths each year. Nosocomial infections can cause severe pneumonia and infections of the urinary tract, bloodstream and other parts of the body. Many types are difficult to attack with antibiotics, and antibiotic resistance is spreading to Gram-negative bacteria that can infect people outside the hospital.
In Gram-negative bacteria, lipopolysaccharides (LPS) and outer-membrane proteins are the major antigenic parts of the bacterial envelope. LPS based vaccines have been extensively studied in the 1970s (Priebe G & Pier G., Vaccines for Pseudomonas aeruginosa 2003. New Bacterial vaccines, edited by Elfis R W, Brodeur B. 260-82). Parke Davis produced a vaccine Pseudogen from LPS of 7 different serogroups. Some activity was observed with Pseudogen in non-randomized trials in cancer and burn patients but not in cystic fibrosis (CF) and leukemia patients. Being LPS based Pseudogen was very toxic and therefore not registered (Priebe, supra). Using two different versions of recombinant fusion proteins of Opr's F and I, von Specht and colleagues have shown that active immunization can protect neutropenic mice and passive immunization can protect SCID mice, both against a challenge dose 1000-fold above the LD50 (von Specht, BU et al., Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant Pseudomonas aeruginosa outer membrane protein F and Outer membrane protein I fusion proteins. Infect Immun 1995; 63(5):1855-1862; Knapp B et al., A recombinant hybrid outer membrane protein for vaccination against Pseudomonas aeruginosa. Vaccine 1999; 17(13-14):1663-1666). Said fusion protein was then tested for safety and immunogenicity in healthy volunteers reaching high levels of specific serum antibodies. To achieve an enhanced mucosal immunogenicity in cystic fibrosis an emulgel formulation of said fusion protein was developed and tested for safety and immunogenicity in healthy volunteers and lung impaired patients. However, the serum antibody response was comparatively low. A systemic i. m. booster has enhanced serum antibody response as compared to solely mucosal vaccination schedule.
An outer membrane protein preparation composed of 4 different strains of Pseudomonas aeruginosa with a molecular weight range of 10-100 kDa was developed as a vaccine in Korea. The vaccine contained minimal amounts of polysaccharide and was tested in a double-blind, placebo-controlled trial in burn patients (Jang I I et al., Human immune response to a Pseudomonas aeruginosa outer membrane protein vaccine. Vaccine 1999; 17(2): 158-68). Antibody levels to the vaccine antigens rose by 2.3-fold in the placebo group (19 patients) and 4.9 fold in the vaccine group (76 patients) (Kim D K et al., Comparison of two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins vaccine in burn patients. Vaccine 2001; 19(9-10):1274-83). Priebe and Pier criticized the study because the follow-up of patients in the trial was incomplete, analysis was not by intention-to-treat, and there were no data regarding clinical outcomes (Priebe, supra). A similar Opr vaccine was tested in Russia 10 years earlier (Stanislaysky E S et al., Clinico-immunological trials of Pseudomonas aeruginosa vaccine. Vaccine 1991; 9(7):491-4). Pseudomonas aeruginosa vaccine (PV) containing predominantly cell-wall protein protective antigens was tested for safety and immunogenicity by immunization of 119 volunteers. The PV vaccine was well tolerated. A high level of specific antibodies persisted for the 5-month period of observation. The antibody titers increased in 94-97% of volunteers and moreover in 45.6% the antibody titers (the number of ELISA units) increased 2.5-3-fold and more. Anti-Pseudomonas aeruginosa plasma was used for the treatment of 46 patients with severe forms of Pseudomonas aeruginosa infection (40 adults and six infants aged up to 2 years) and 87% of the patients recovered. There have been no follow-up studies with the PV vaccine after 1991.
Hospital-acquired infections are one of the major causes of death and serious illness worldwide, resulting in an annual cost burden of more than USD 20 billion in the developed world. In the United States and Europe about 6 million patients become infected annually resulting in 140,000 deaths per year. The incidence of nosocomial infections is steadily increasing due to increasing medical interventions and antibiotic resistance. Thus, minimizing risk of mortality through hospital acquired infections by e.g. vaccination of burn victims and fibrosis patients, ICU patients and ventilated ICU patients is and is expected to become even more so a major unmet medical need in said patients.
It has recently been found (U.S. provisional application with application No. 61/426,760) that a vaccine of the above-described hybrid fusion protein comprising the Pseudomonas aeruginosa outer membrane protein I (OprI or OMPI) which is fused with its amino terminal end to the carboxy-terminal end of a carboxy-terminal portion of the Pseudomonas aeruginosa outer membrane protein F (OprF or OMPF) reduced the mortality rate in mechanically ventilated intensive care patients significantly over alum as placebo control. Mechanically ventilated intensive care patients are at particular risk of acquiring severe and often life-threatening forms of Pseudomonas aeruginosa or other infections, such as Ventilator-Associated Pneumonia (VAP), sepsis or soft tissue infection. Such infections also may affect burn victims, severely burned victims, cancer and transplant patients who are immunosuppressed, and cystic fibrosis patients, Intensive Care Unit (ICU) patients or generally all hospitalized patients.
Generally, the expression of soluble OprF/I fusion protein in E. coli leads to the formation of non immunological aggregates and misfolded variants. According to Worgall et al. (Worgall S et al., Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in The Capsid., J. of Clinical Investigation, 2005, 115(5), 1281-1289) it is assumed that the native OprF protein has one disulphide bridge from Cys200 to Cys209 of SEQ ID NO: 1 and two free cysteines at Cys215 and Cys229 of SEQ ID NO: 1. In another publication (Rawling E G et al., Epitope Mapping of the Pseudomonas aeruginosa Major Outer membrane Protein OprF., Infection and Immunity, 1995, 63 (1), 38-42), however, two disulphide bonds from Cys200 to Cys209 and from Cys215 to Cys229 of SEQ ID NO: 1 are proposed. It cannot be expected that the reported disulphide bond pairing applies to the fusion protein OprF/I since only amino acid No. 190 to amino acid No. 342 of SEQ ID NO: 1 from the native OprF protein are expressed. Since native OprF is an outer membrane protein and contains several transmembrane spans, it is expected that folding in an aqueous environment differs from the folded structure of the natively expressed protein located in a membrane.
In addition, a pharmaceutical composition should be homogenous and stable. Thus, both good manufacturing practice as well as regulatory authority guidelines require that a dosage form of a pharmaceutical or pharmaceutical combination should be in the form of a homogeneous dispersion with respect to the active substances. There is a concern in the field regarding aggregates and a potential for immunogenicity (Leonard J. Schiff, Biotechnology Products Derived from Mammalian Cell Lines: Impact of Manufacturing Changes (2004) Regulatory Affairs Focus, October 2004, pages 29-31).